Method for treatment of cancer and infectious diseases and compositions useful in same

ABSTRACT

Administration of expressible polynucleolides encoding eukaryotic heat shock proteins to mammalian cells leads to the stimulation of an immune response to antigens present in those cells. This makes it possible to stimulate an immune response to target antigens, including target tumor antigens or antigens associated with an infectious disease, without having to isolate a unique antigen or antigen-associated heat shock protein for each target antigen by administering to a mammalian subject or to a group of mammalian cells containing the antigen, an expressible polynucleotide encoding a heat shock protein. The expressed heat shock protein may have the same structure as native heat shock proteins, or may have a modified form adapted to control the trafficking of the expressed heat shock protein within the cells.

[0001] The invention described herein was made in the course of workunder NIH Core Grant No. CA 08748. The United States government may havecertain rights in this invention.

BACKGROUND OF THE INVENTION

[0002] This application relates to the use of heat shock proteins andsimilar peptide-binding proteins to stimulate an immunological responseagainst antigens, including cancer-related antigens, autoimmune antigensand infectious disease antigens, found in a mammalian host.

[0003] Heat shock proteins were originally observed to be expressed inincreased amounts in mammalian cells which were exposed to suddenelevations of temperature, while the expression of most cellularproteins is significantly reduced. It has since been determined thatsuch proteins are produced in response to various types of stress,including glucose deprivation. As used herein, the term “heat shockprotein” will be used to encompass both proteins that are expresslylabeled as such as well as other stress proteins, including homologs ofsuch proteins that are expressed constitutively (i.e., in the absence ofstressful conditions). Examples of heat shock proteins include BiP (alsoreferred to as grp78), hsp/hsc70, gp96 (grp94), hsp60, hsp40 and hsp90.

[0004] Heat shock proteins have the ability to bind other proteins intheir non-native states, and in particular to bind nascent proteinsemerging from the ribosomes or extruded into endoplasmic reticulum.Hendrick and Hartl, Ann. Rev. Biochem. 62: 349-384 (1993); Hartl, Nature381: 571-580 (1996). Further, heat shock proteins have been shown toplay an important role in the proper folding and assembly of proteins inthe cytosol, endoplasmic reticulum and mitochondria; in view of thisfunction, they are referred to as “molecular chaperones”. Frydman etal., Nature 370: 111-117 (1994); Hendrick and Hartl., Ann. Rev. Biochem.62:349-384 (1993); Hartl., Nature 381:571-580 (1996)

[0005] For example, the protein BiP, a member of a class of heat shockproteins referred to as the hsp70 family, has been found to bind tonewly synthesized unfolded μ immuno-globulin heavy chain prior to itsassembly with light chain in the endoplasmic reticulum. Hendershot etal., J. Cell Biol. 104:761-767 (1987). Another heat shock protein, gp96,is a member of the hsp90 family of stress proteins which localize in theendoplasmic reticulum. Li and Srivastava, EMBO J. 12:3143-3151 (1993);Mazzarella and Green, J. Biol. Chem. 262:8875-8883 (1987). It has beenproposed that gp96 may assist in the assembly of multi-subunit proteinsin the endoplasmic reticulum. Wiech et al., Nature 358:169-170 (1992).

[0006] It has been observed that heat shock proteins prepared fromtumors in experimental animals were able to induce immune responses in atumor-specific manner; that is to say, heat shock protein purified froma particular tumor could induce an immune response in an experimentalanimal which would inhibit the growth of the same tumor, but not othertumors. Srivastava and Maki, 1991, Curr. Topics Microbiol. 167: 109-123(1991). The source of the tumor-specific immunogenicity has not beenconfirmed. Genes encoding heat shock proteins have not been found toexhibit tumor-specific DNA polymorphism. Srivastava and Udono, Curr.Opin. Immunol. 6:728-732 (1994). High resolution gel electrophoresis hasindicated that gp96 may be heterogeneous at the molecular level. Feldwegand Srivastava, Int. J. Cancer 63:310-314 (1995). Evidence suggests thatthe source of heterogeneity may be populations of small peptidesadherent to the heat shock protein, which may number in the hundreds.Id. It has been proposed that a wide diversity of peptides adherent totumor-synthesized heat shock proteins may render such proteins capableof eliciting an immune response in subjects having diverse HLAphenotypes, in contrast to more traditional immunogens which may besomewhat HLA-restricted in their efficacy. Id.

[0007] Lukacs et al., J. Exp. Med. 178:343-348 (1993). have reported thetransfection of tumor cells with a mycobacterial heat shockprotein-encoding gene, and the observation that the transfected cellslose tumorigenicity and induce what appears to be T-cell mediatedprotection against tumors in mice immunized using the transfected cells.Lukacs et al. suggest that the loss of tumorigenicity could result fromthe interaction of the heat shock protein with p53 via increasedefficiency of chaperone activity to produce proper folding andconformation of otherwise ineffective p53 protein. They further suggestthat the highly immunogenic nature of the 65 kD bacterial hsp enhancesthe recognition of other, tumor-associated antigen molecules.

[0008] It has been suggested in the literature that mycobacterial heatshock proteins may play a role in the onset of autoimmune diseases sucha rheumatoid arthritis. Thus, the practical utility of such bacterialproteins in vaccines for the treatment of humans is questionable. It isan object of the present invention to provide vaccine compositions whichcan be used to stimulate an immune response to antigens, including tumorand infectious disease antigens, present in mammalian cells without theintroduction of mycobacterial proteins.

SUMMARY OF THE INVENTION

[0009] It has now been found that administration of expressiblepolynucleotides encoding eukaryotic heat shock proteins to mammaliancells leads to the stimulation of an immune response to antigens presentin those cells. This makes it possible to stimulate an immune responseto treat a subject's disease condition, including an immune response toa tumor or an infectious disease, without having to isolate orcharacterize an antigen associated with the disease. Thus, the presentinvention provides a method for stimulating a therapeutic orprophylactic immune response in a mammalian subject by treating thesubject or a group of cells from the subject with an expressiblepolynucleotide encoding a eukaryotic heat shock protein. The expressedheat shock protein may have the same structure as native heat shockproteins, or may be a modified form adapted to control the traffickingof the expressed heat shock protein within the cells.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] FIGS. 1A-H show tumor formation in mice injected with CMS-5sarcoma cells and CMS-5 sarcoma cells acutely transfected withBiP-expressing vectors; and

[0011] FIGS. 2A-F show tumor formation in mice injected with CMS-5sarcoma cells and CMS-5 sarcoma cells stably transfected withBiP-expressing vectors.

DETAILED DESCRIPTION OF THE INVENTION

[0012] The present invention provides a method for inducing or enhancingan immune response to an antigen present in a target group of cellswithout requiring prior identification, characterization or isolation ofthe antigen. The method of the invention may be used to induce an immuneresponse to the antigen in the case where there is no existingdetectable disease-related immune response prior to practicing themethod, or to enhance the disease-related immune response to a greaterlevel. For purposes of simplicity, the specification and claims of thisapplication will use the term stimulating an immune response toencompass both inducing a new immune response and enhancing apre-existing immune response.

[0013] We have found that administration of expressible polynucleotidesencoding eukaryotic heat shock proteins to mammalian cells leads to thestimulation of an immune response to antigens present in those cells.While we do not intend to be bound by any particular mechanism, the fullpotential of our observations is best understood in the context of twomechanistic models which explain the observations.

[0014] The first mechanism is based on the fact that heat shock proteinslike BiP and gp96 are normally localized in the endoplasmic reticulumand are known to bind peptides and proteins in this location, and thebelief that such binding may be a step in the presentation of an antigento the immune system. Increasing the concentration of one or more heatshock proteins resident in the endoplasmic reticulum may cause animprovement in the efficiency of this process, and thus an increasedimmune response to the antigens present in the transfected cells.

[0015] The second mechanism is based upon the understanding that heatshock proteins and other proteins secreted from the endoplasmicreticulum may be recaptured by receptors known as erd-2 (KDEL) receptorsand returned to the endoplasmic reticulum. This recapture processrequires the presence of a specific retention sequence on the heat shockprotein such XDEL. XDEL is the single letter amino-acid code for theamino acid sequence variable-Asp-Glu-Leu. Specific carboxy-terminalretention sequences are KDEL (Lys-Asp-Glu-Leu) and HDEL(His-Asp-Glu-Leu). By eliminating this retention sequence in heat shockproteins which bind to antigens or by otherwise interfering with theability of the erd-2 receptor to recapture these heat shock proteins,the amount of antigen-associated heat shock protein secreted by a celland therefore accessible to the immune system can be increased.

[0016] In accordance with either of these mechanisms, the stimulation ofthe immune response which is observed when an expressible polynucleotideencoding a heat shock protein is expressed in the cell flows from anincrease in the amount of antigen-associated heat shock protein. Thus,in accordance with one embodiment of the invention there is provided amethod for stimulating an immune response to an antigen present in atarget group of mammalian cells, comprising administering to the targetgroup of cells a composition effective to increase the amount ofantigen-associated heat shock protein present in the cells. This can beaccomplished in several ways.

[0017] First, the amount of antigen-associated heat shock protein can beincreased by administering a polynucleotide encoding a heat shockprotein which is expressed to produce recombinant heat shock proteinthat is processed and transported within the cell in the same manner aswild-type heat shock protein.

[0018] Second, the amount of antigen-associated heat shock protein canbe increased by administering an expressible polynucleotide encoding aheat shock protein in a modified form adapted to control the traffickingof the expressed heat shock protein within the cells. This would includealterations of heat shock protein which either facilitate its secretionfrom the cell or its localization and/or retention on the cell. Forexample, secretion may be facilitated by mutating or eliminatingportions of the heat shock protein that serve to retain the heat shockprotein in the cell (for example by deleting sequences recognized by theerd-2 receptor such as KDEL or a functionally equivalent sequence or byadding an agent that interferes with binding of heat shock protein toerd-2), or by supplying mutated non-heat shock proteins (e.g. erd-2mutants) that increase heat shock protein secretion. Alternativelylocalization and retention may be accomplished by increasing the numberand/or strength of retention or retrieval signals (e.g. increasing erd-2levels) or my by adding to a heat shock protein, a membrane anchorcontaining additional retention or retrieval sequence as well astransmembrane and cytoplasmic domains (e.g., KKXX at the C-terminal end,or XXRR at the N-terminal end or function equivalents thereof.)

[0019] Third, the amount of an antigen-associated heat shock protein canbe increased by administering an expressible polynucleotide encoding apeptide or peptide which competitively inhibits the interaction of heatshock proteins with cellular elements to modify the trafficking of thoseheat shock proteins within the cell.

Preparation of Expressible Polynucleotides

[0020] The construction of expressible polynucleotides for use in thethree embodiments of the invention share many common elements and usegenerally well known techniques. These techniques are set forth innumerous literature references known to persons skilled in the art andso will not be repeated exhaustively here.

[0021] Expressible polynucleotide encoding a heat shock protein which isexpressed to produce recombinant heat shock protein that is processedand transported within the cell in the same manner as wild-type heatshock protein can be constructed by incorporating a cDNA encoding awild-type heat shock protein or a modified heat shock protein thatretains both its heat shock protein functionality and the XDEL retentionsequence into a mammalian expression vector. Genes for various mammalianheat shock proteins have been cloned and sequenced, including, but notlimited to, gp96, human: Genebank Accession No. X15187; Maki et al.,Proc. Nat'l Acad. Sci. 87: 5658-5562 (1990), mouse: Genebank AccessionNo. M16370; Srivastava et al., Proc. Nat'l Acad. Sci. 84:3807-3811(1987)); BiP, mouse: Genebank Accession No. U16277; Haas et al., Proc.Nat'l Acad. Sci. U.S.A. 85: 2250-2254 (1988), human: Genebank AccessionNo. M19645; Ting et al., DNA 7: 275-286 (1988); hsp70, mouse: GenebankAccession No. M35021; Hunt et al., Gene 87: 199-204 (1990), human:Genebank Accession No. M24743; Hunt et al., Proc. Nat'l Acad. Sci.U.S.A. 82: 6455-6489 (1995); and hsp40human: Genebank Accession No.D49547; Ohtsuka K., Biochem Biophys. Res. Commun. 197: 235-240 (1993).

[0022] In general, any type of mammalian expression vector can be used,although those with the highest transfection and expression efficienciesare preferred to maximize the levels of expression. Specific types ofvectors which can be employed include herpes simplex viral basedvectors: pHSV1 (Geller et al. Proc. Natl. Acad. Sci 87:8950-8954(1990)); recombinant retroviral vectors: MFG (Jaffee et al. Cancer Res.53:2221-2226 (1993)); Moloney-based retroviral vectors: LN, LNSX, LNCX,LXSN (Miller and Rosman Biotechniques 7:980-989 (1989)); vaccinia viralvector: MVA (Sutter and Moss Proc. Natl. Acad. Sci. 89:10847-10851(1992)); recombinant adenovirus vectors pJM17 (Ali et al Gene Therapy1:367-384 (1994)), (Berkner K. L. Biotechniques 6:616-624 1988); secondgeneration adenovirus vector: DE1/DE4 adenoviral vectors (Wang and FinerNature Medicine 2:714-716 (1996) ); and Adeno-associated viral vectors:AAV/Neo (Muro-Cacho et al. J. Immunotherapy 11:231-237 (1992)). Specificsuitable expression systems for this purpose include pCDNA3(In-Vitrogen), plasmid AH5 (which contains the SV40 origin and theadenovirus major late promoter), pRC/CMV (In-Vitrogen), pCMU II (Paaboet al., EMBO J. 5: 1921-1927 (1986)), pZip-Neo SV (Cepko et al., Cell37: 1053-1062 (1984)) and pSRα (DNAX, Palo Alto, Calif.).

[0023] Expressible polynucleotides encoding any of the various modifiedforms heat shock proteins described above are constructed using similartechniques. For example, heat shock proteins can be produced which aremodified to delete or block the carboxy-terminal XDEL sequence. Such aheat shock protein is adapted to permit the heat shock protein to besecreted from the endoplasmic reticulum and to interfere with the returnof the protein to the endoplasmic reticulum that is mediated by theerd-2 receptors because the signal peptide recognized by the erd-2receptors is missing. Proteins of this type are referred to in thespecification and claims of this application as XDEL-negative heat shockproteins.

[0024] Expressible XDEL-negative polynucleotides can be constructed bydeleting the nucleotides encoding the amino acid sequence XDEL at thecarboxy-terminus of the heat shock protein. This can be accomplished byPCR amplification of the wild-type cDNA using a primer that hybridizesin the region immediately adjacent to the portion coding for the XDELsignal, combined with a restriction site to be used for cloning theproduct into a desired vector for expression of the modified heat shockprotein in mammalian cells as described above.

[0025] As an alternative to the use of expressible polynucleotidesencoding deletion mutants of heat shock proteins, the method of theinvention may also employ expressible polynucleotides which encode heatshock proteins in which the carboxy-terminal retention sequence XDEL ismasked by the addition of additional amino acids to the carboxy-terminalend. The masking amino acids can serve only the masking function, or canbe derived from an infectious agent (for example E7/E6 from humanpapilloma virus) in which case the masking amino acids may also functionas an antigen.

[0026] Addition of masking amino acids can be accomplished as describedin Munro et al., Cell 48: 899-907 (1987) in which a mutant (SAGGL)having two amino acids added after the XDEL was prepared by cloning inappropriate restriction sites to permit substitution of a sequenceincluding the additional bases, or by PCR amplification of the heatshock protein using a primer that hybridizes with the bases encoding theXDEL retention sequence and which has additional bases encoding theadded nucleotides inserted between the XDEL-encoding bases and the stopcodon. The number of additional amino acids does not matter, so long asthe added amino acids do not alter the function of the heat shockprotein. The nucleotides encoding the masked heat shock protein are thenintroduced into a mammalian expression vector as described above.

[0027] Another method which may be used to increase the amount ofantigen-associated heat shock protein in the cells is to modifycytosolic heat shock proteins with an amino-terminal signal which causesthe protein to be taken up into the endoplasmic reticulum where it cangain access to the secretory pathway. Amino-terminal signals which canaccomplish this function are described in the art, including for examplein von Heijne, G., J. Mol. Biol. 184: 99-105 (1985). These signalsgenerally comprise a positively-charged amino acid or group of a fewamino acids at the amino terminal end of the peptide, a hydrophobic coreregion and a third region of greater polarity than the hydrophobicregion. Such additional signal sequences can be introduced using PCRwith a modified primer that includes the bases for the desiredamino-terminal amino acids. A representative cytosolic heat shockprotein which might be modified in this manner is hsp 70.

[0028] The expressible polynucleotide used in the method of the presentinvention may also encode a peptide or protein that competitivelyinhibits the interaction of heat shock proteins with cellular elementsto modify the trafficking of those heat shock proteins within the cell.In particular, the expressible polynucleotide may encode a peptide orprotein that includes a carboxy-terminal XDEL retention sequence whichwill compete with heat shock proteins, including endogenous heat shockproteins, for binding to the erd-2 receptors. This competitive peptidemay itself be a heat shock protein, for example BiP, that retains theXDEL retention sequence, or it may be some other peptide or protein witha carboxy-terminal XDEL sequence.

Administration of Expressible Polynucleotides

[0029] The resulting expressible polynucleotide is delivered into cellsof the subject by ex vivo or in vivo methods, for example as part of aviral vector as described above, or as naked DNA. Suitable methodsinclude injection directly into tissue and tumors, transfection usingliposomes (Fraley et al, Nature 370: 111-117 (1980)), receptor-mediatedendocytosis (Zatloukal, et al., Ann. N.Y. Acad. Sci. 660: 136-153(1992)), particle bombardment-mediated gene transfer (Eisenbraun et al.,DNA & Cell. Biol. 12: 791-797 (1993)) and transfection using peptidepresenting bacteriophage. Barry et al. Nature Medicine 2: 299-305(1996).

[0030] The expressible polynucleotide may be administered to a subjectin need of treatment in order to obtain a therapeutic immune response.The term “therapeutic immune response”, as used herein, refers to anincrease in humoral and/or cellular immunity, as measured by standardtechniques, which is directed toward an antigen associated with thesubject's disease condition. Preferably, but not by way of limitation,the induced level of humoral immunity directed toward the antigen is atleast four-fold, and preferably at least 16-fold greater than the levelsof the humoral immunity directed toward the antigen prior to theadministration of the expressible polynucleotide of this invention tothe subject. The immune response may also be measured qualitatively, bymeans of a suitable in vitro assay or in vivo, wherein an arrest inprogression or a remission of neoplastic or infectious disease in thesubject is considered to indicate the induction of a therapeutic immuneresponse.

[0031] A further aspect of the invention is the administration of anexpressible polynucleotide in combination with other forms of treatmentincluding surgery, radiation therapy, and chemotherapy, to provide avaccination against recurrence of cancer. By priming the immune systemof subjects to recognize the cancer cells of their own cancers, theimmune system will be more prepared to counter a subsequent regrowth,thus improving the prognosis for the subjects. This same type ofvaccination can be used to improve a subject's resistance to recurrentdiseases, including many parasitic and viral diseases. Thus, theexpressible polynucleotides of the invention may be administered tostimulate a prophylactic immune response. The term “prophylactic immuneresponse”, as used herein, refers to an increase in long term humoraland/or cellular immunity, as measured by standard techniques, which isdirected toward an antigen associated with the subject's diseasecondition. In general, the prophylactic immune response is measuredqualitatively, wherein a delay in the onset of recurring system isachieved.

[0032] The subject treated in the method of the invention may be a humanor non-human subject.

[0033] In various embodiments of the invention, the polynucleotide mayencode a heat shock protein derived from the same species oralternatively from a different species relative to the species of thesubject.

[0034] The cells into which the expressible polynucleotide areintroduced may be neoplastic cells or other cancer cells from thesubject to be treated or vaccinated. In particular, the method of theinvention is usefully applied to treat or vaccinate subjects sufferingfrom cancer including solid tumors and neoplastic disease such assarcoma, lymphoma, carcinoma, leukemia and melanoma. The method of theinvention may also be utilized to stimulate an immune response toinfectious diseases, including parasitic, fungal, yeast, bacterial,mycoplasmal and viral diseases, where a particular class of cells can beidentified as harboring the infective entity. For example, but not byway of limitation, the cells treated may be infected with a humanpapilloma virus, a herpes virus such as herpes simplex or herpes zoster,a retrovirus such as human immunodeficiency virus 1 or 2, a hepatitisvirus, an influenza virus, a rhinovirus, respiratory syncytial virus,cytomegalovirus, adenovirus, Mycoplasma pneumoniae, a bacterium of thegenus Salmonella, Staphylococcus, Streptococcus, Enterococcus,Clostridium, Escherichia, Klebsiella, Vibrio, Mycobacterium, amoeba, amalarial parasite, Trypanosoma cruzi, etc. Thus, for example, in thecase of human papilloma virus (HPV), the expressible polynucleotidecould be introduced into epithelial cells infected with HPV from thesubject to be treated.

[0035] The method of the invention can be practiced using variouscompositions. One such composition of matter is a recombinant vectorcomprising a promoter system effective to promote expression of thevector in mammalian cells; and a region encoding a XDEL-negative heatshock protein. The vector may be one in which the bases encoding aXDEL-retention sequence have been deleted from the region encoding theheat shock protein, or one in which additional bases have been added tothe region encoding the heat shock protein to mask the XDEL-retentionsequence in the expressed product. An exemplary vector would includegp96, preferably human gp96, as the heat stock protein.

[0036] A further composition of matter in accordance with the inventionis a recombinant vector comprising a promoter system effective topromote expression of the vector in mammalian cells; and a regionencoding a cytosolic heat shock protein such as hsp70to which a signalcomprising a positively-charged amino acid or group of a few amino acidsat the amino terminal end of the peptide, a hydrophobic core region anda third region of greater polarity than the hydrophobic region isattached to promote uptake of the expressed cytosolic heat shock proteinby the endoplasmic reticulum has been added.

[0037] The invention will now be further described by way of thefollowing non-limiting examples.

EXAMPLE 1 Preparation of Xdel Deletion Mutants

[0038] The preparation of mammalian expression vectors encodingfull-length and KDEL-deleted Drosophila BiP cDNAs (dBiP, also referredto a grp78) is described in Munro et al., Cell 48: 899-907 (1987). Theexpression vector encoding dBIP with a KDEL deletion (KDEL-deleted dBiP,SAGM2) has the KDEL retention sequence replaced with a c-myc sequence ina modified AHP2 plasmid. A further construct, WT dBiP (SAGMK1) has thec-myc sequence inserted between the KDEL retention sequence and thebalance of the dBiP.

[0039] Wild type mouse BiP cDNA was cloned into plasmid pBSBiP. Haas,Proc. Nat'l Acad. Sci. USA 85: 2250-2254 (1988) In this plasmid, the BiPsequence is flanked by BamH I sites. The BamH I fragment was thereforerecovered from pBSBiP and cloned into the BamH I site of mammalianexpression vector pCDNA3 (In-Vitrogen). Mouse KDEL-deleted BiP wasconstructed using pCDNA3 by cloning the Bgl II-EcoR V PCR fragment(nucleotide 1360 to nucleotide 1981) of BiP using two oligonucleotideprimers complementary to BiP. The nucleotides coding for the last fouramino acids (KDEL) were omitted, and a stop codon and an EcoR Vrestriction site put in its place.

EXAMPLE 2 Preparation of Tumor Cells Expressing BiP

[0040] CMS-5 sarcoma cells, a methylocholanthrene-induced fibrosarcomaof BALB/c mouse origin, were adapted to culture and grown in DMEM medium(Gibco Life Technologies, Inc.) supplemented with 10% FCS. Sub-confluentmonolayers (2×10⁹ cells) were transfected with 2 ug of mammalianexpression vector containing BiP or KDEL-deleted BiP using lipofectamine(Gibco) according to the manufacturer's directions. Briefly, theexpression vector and 6 ul of lipofectamine were diluted separately in100 uL serum-free medium (OPTI-MEDM® I Reduced Serum Medium, Gibco-BRL).The two solutions were then mixed and incubated at room temperature for45 minutes to allow formation of DNA-liposome complexes. 800 uLOPTI-MEM° was added to the complexes, mixed, and overlaid onto rinsedcells. After a 6 hour incubation at 37° C., 1 mL growth mediumcontaining 20% FCS was added. Fresh medium was added to the cells 24hours post-transfection.

[0041] Stable clones were selected by adding 800 ug/mL geneticin(Gibco-BRL) to the cells 72 hours later. The selection medium waschanged every 3 days. Colonies of stably transfected cells were seenafter 10 to 14 days. Expression of BiP for each clone picked was assayedfor by radiolabeling. Newly synthesized BiP was detectable byimmunoprecipitation and gel electrophoresis.

[0042] Acutely (transiently) BiP expressing clones were trypsinized andused for animal injection 60 hours post-transfection.

EXAMPLE 3 Vaccination with BiP Expressing Tumor Cells

[0043] Freshly prepared acutely transfected CMS-S sarcoma cells preparedin accordance with Example 2 were prepared for use in vaccination ofmice by trypsinization and then washing three times in PBS. 2×10⁶ cellswere injected intradermally into the abdomen of CB6F-1/J[Balb/c×C57BL/6F1] mice (Jackson Laboratories) and monitored for tumorgrowth over a 90 day period. The results are summarized in Table 1 andshown graphically in FIGS. 1A-H. As shown, injection with the parentalCMS-5 sarcoma cells, with mock transfected cells, and with cellstransfected with KDEL-deleted vectors led in all cases to the formationof tumors. Tumors also developed in one control mouse where the cellswere transfected with the vector only (no BiP) . On the other hand, onlyone of six mice transfected with the complete wild-type drosophila BiP(including the c-myc) or wild-type mouse BiP developed tumors. TABLE 1Number of Tumor Free Mice per Percent number of mice Tumor Free CellLine injected Survivals Parental CMS5 0/4  0% Mock Transfected 0/3  0%Vector Only 1/3  33% Mouse BiP wild type 2/3  66% Mouse BiP KDEL deleted0/3  0% Drosophila BiP wild type 3/3 100% Drosophila BiP KDEL deleted0/3  0%

EXAMPLE 4

[0044] The experiment of Example 3 was repeated using larger groups ofmice and using two different clones of stable mouse BiP-expressingtransfected cells. As shown in Table 2 and FIGS. 2A-F, the results wereessentially the same, with none of the mice injected with the vectorexpressing wild-type mouse BiP having any detected tumor formation.Number of Tumor Free mice per Percent number mice Tumor Free Cell Linesinjected Survivals Parental CMS5 0/4  0% Vector Only  4/15  27% MouseBiP wild type: clone 1 6/6 100% Mouse BiP wild type: clone 2 11/11 100%Mouse BiP KDEL deleted: clone 1 1/9  11% Mouse BiP KDEL deleted: clone 2 2/15  13%

EXAMPLE 5

[0045] Tumor-free mice that had been injected with the wild-type BiPexpression vector in the experiment of Example 4 were rechallenged afteran interval of two months by injection of 2×10⁶ or 10×10⁶ CMS-5 sarcomacells into the left flank of each mouse. Unimmunized CB6F-1/J mice ofthe same age group that had not been previously immunized were injectedat the same time with 2×10⁶ CMS-5 sarcoma cells as controls. As shown inTable 3, none of the previously immunized mice developed detectabletumors, while all three of the control mice developed tumors and died.TABLE 3 Number of Tumor Free Mice/Number of Mice Rejected Tumor Injected% Survivals Mouse Bip wild type: 3/3 100% clone 1 3/3 100% Mouse BiPwild type: 5/5 100% clone 2 6/6 100% Control 0/3  0%

EXAMPLE 6 Construction of Vector Expressing gp96

[0046] To create an expressible vector encoding gp96, the coding regionof murine GP96 (mGP96) was digested with BamH I, blunted with Klenow andcloned into the mammalian expression vector pRC/CMV (In-Vitrogen) whichhas been digested with Xba I and also blunted with Klenow. The resultingplasmid, p96/CMV was then used as template for generating a fragmentspanning nucleotides 1653 to 2482 that lacks the nucleotides encodingthe amino acids KDEL. The primers used for amplification of thisfragment were 5′ primer: GCGGATCCTAGTTTAGACCAGTATGTC (SEQ ID No. 1)3′ primer: CCGAATTCGGGCCCCAATTTACTCTGTAGATTCCTTTTCTGTTT (SEQ ID No. 2)

[0047] The restrictions sites for BamH I in the 5′-primer and EcoR I andSal I in the 3′-primer are shown in bold italics. A stop codon (shownunderlined) is provided four nucleotides from the Sal I restriction sitein the 3′-primer. The BamH I/EcoR I PCR fragment was subcloned intopBlueScript II (Stratagene) to further amplify the amount of DNA. Theamplified DNA was then digested with PflM I and Apa I, gel purified andsubcloned into p96/CMV to give pMS215 which codes for KDEL-deleted gp96.

EXAMPLE 7 Vaccination with Gp96 Expressing Vectors

[0048] Plasmids encoding the wt mGp96 (pMS216) and KDEL deleted mGp96(215) are transfected into CMS-5 sarcoma cells as described in Example2. 86 h post transfection, the acutely transfected CMS-5 sarcoma cellsare trypsinized, washed in PBS and prepared for use in vaccination ofmice as described in Example 3.

[0049] Various publications are cited herein, the contents of which arehereby incorporated by reference in their entireties.

1 2 27 nucleic acid double linear Genomic DNA no yes internal mouseamplification primer for gp96 1 GCGGATCCTA GTTTAGACCA GTATGTC 27 44nucleic acid double linear Genomic DNA no no internal mouseamplification primer for gp96 2 CCGAATTCGG GCCCCAATTT ACTCTGTAGATTCCTTTTCT GTTT 44

1. A method for stimulating a therapeutic immune response in a subjectin need of such treatment, comprising the step of introducing to thesubject, or to a target group of cells from the subject, an expressiblepolynucleotide encoding a eukaryotic heat shock protein.
 2. A method forstimulating a prophylactic immune response against recurrence of adisease from which a subject is suffering, comprising administering tothe subject, or to a target group of cells from the subject harboring anantigen associated with the disease, an expressible polynucleotideencoding a eukaryotic heat shock protein.
 3. The method according toclaim 1 or 2, wherein the eukaryotic heat shock protein is anXDEL-negative heat shock protein.
 4. The method according to claim 1 or2, wherein the eukaryotic heat shock protein is a heat shock proteinfrom which the carboxy-terminal XDEL retention sequence has beendeleted.
 5. The method according to claim 1 or 2, wherein the eukaryoticheat shock protein is a heat shock protein in which the carboxy-terminalXDEL retention sequence has been masked.
 6. The method according toclaim 1 or 2, wherein the expressible polyhucleotide encodes aeukaryotic heat shock protein having a carboxy-terminal retentionsequence of the sequence XDEL.
 7. The method according to claim 6,wherein the eukaryotic heat shock protein is BiP.
 9. The methodaccording to any of claims 1 to 8, wherein the cells are cancer cells.10. The method according to any of claims 1 to 8, wherein the cells areneoplastic cells.
 11. The method according to claim 10, wherein theneoplastic cells are selected from among sarcoma cells, lymphoma cells,leukemia cells, carcinoma cells and melanoma cells.
 12. The methodaccording to any of claims 1 to 8, wherein the cells are infected with avirus.
 13. The method according to any of claims 1 to 8, wherein thecells are infected with a parasite.
 14. The method according to any ofclaims 1 to 8, wherein the cells are infected with a mycoplasma.
 15. Themethod according to any of claims 1 to 8, wherein the cells are infectedwith a bacterium.
 16. The method according to any of claims 1 to 8,wherein the cells are infected with a fungus or yeast.
 17. The methodaccording to any of claim 1 to 16, wherein the expressiblepolynucleotide is introduced into a target group of cells ex vivo, andthe target group of cells are thereafter administered to the subject.18. The method according to claim 1, wherein the composition isadministered by transfection in a liposome.
 19. The method according toany of claim 1 to 18, wherein the subject is a human.
 20. A recombinantvector comprising (a) a promoter system effective to promote expressionof the vector in mammalian cells; and (b) a region encoding a cytosolicheat shock protein (i) an amino terminal signal sequence effective topromote uptake of the expressed cytosolic heat shock protein by theendoplasmic reticulum and (ii) a carboxy-terminal retention sequenceeffective to promote retention of the heat shock protein in theendoplasmic reticulum.
 21. The recombinant vector of claim 20, whereinthe signal sequence comprises a positively charged N-terminal region, ahydrophobic core region and a third region of greater polarity than thehydrophobic region.
 22. The recombinant vector according to claim 20 or21, wherein the cytosolic heat shock protein is hsp70.
 23. A method forstimulating an immune response to an antigen present in a target groupof mammalian cells, comprising administering to the target group ofcells an expressible polynucleotide, wherein expression of thepolynucleotide is effective to increase the amount of antigen-associatedheat shock protein present in the cells.
 24. The method according toclaim 23, wherein the expressible polynucleotide encodes a eukaryoticheat shock protein.
 25. The method according to claim 23 or 24, whereinthe expressible polynucleotide encodes a peptide having a retentionsequence recognized by the erd-2 receptors.
 26. The method according toclaim 25, wherein the retention sequence is XDEL.